Inward rectifying K (Kir) channels regulate excitability in many tissues, and several diseases result from mutations of Kir channel genes. During previous award periods we demonstrated that soluble cytoplasmic factors cause intrinsic rectification in strong inward rectifiers, identified these factors as polyamines, and confirmed that they act as 'long-pore plugs' to block the channel permeation pathway. Recent crystallization of two different K channel pore structures has illuminated mechanisms of permeation and gating in K channels. However, the structural details of rectification and of gating in Kir channels remain unclear. Preliminary data lead us to specific hypotheses regarding the molecular details of polyamine-induction of rectification and of ligand-gating mechanisms in Kir channels. These hypotheses will be critically examined in three experimental Aims, utilizing a combination of biochemical and biophysical techniques, together with molecular modeling to gain previously unobtainable insight to the mechanisms of inward rectification and gating. The work will provide information that will form the background to the development of rational therapies for cardiac arrhythmias, epilepsy and other disorders of cell excitability through modulation of Kir channel activity.